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EP0323149B1 - Vektoren und Verbindungen zur Expression von Zymogen-Formen von menschlichem Protein C - Google Patents

Vektoren und Verbindungen zur Expression von Zymogen-Formen von menschlichem Protein C Download PDF

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Publication number
EP0323149B1
EP0323149B1 EP88312201A EP88312201A EP0323149B1 EP 0323149 B1 EP0323149 B1 EP 0323149B1 EP 88312201 A EP88312201 A EP 88312201A EP 88312201 A EP88312201 A EP 88312201A EP 0323149 B1 EP0323149 B1 EP 0323149B1
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dna
plasmid
protein
plpc
host cell
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EP0323149A2 (de
EP0323149A3 (en
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Nils Ulrik Bang
Hartmut Josef Ehrlich
Brian William Grinnell
Sau-Chi Betty Yan
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6464—Protein C (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

  • the present invention relates to novel DNA compounds and recombinant DNA cloning vectors that encode novel zymogen forms of human protein C. These zymogens can be activated in vivo by thrombin alone at a rate of clinical significance and are much more susceptible to activation by thrombin/thrombomodulin than native protein C zymogen.
  • the expression vectors provide a simple and efficient means for expressing these human protein C zymogens in recombinant host cells. Native human protein C zymogens require treatment with high levels of thrombin, or thrombin and thrombomodulin, or other expensive enzymes for activation.
  • the present invention provides a method for producing zymogen forms of human protein C that serve as much better substrates for thrombin and consequently can be activated in the presence of lower levels of thrombin, or thrombin/thrombomodulin, or other enzymes.
  • the zymogen forms of human protein C of the invention can be activated by thrombin even in the presence of physiological Ca2+, which is inhibitory to the activation of native protein C zymogen by thrombin.
  • the novel zymogen forms of human protein C differ from those known in the art in the amino acid residue sequence of the activation peptide, which is removed from the zymogen forms to produce activated human protein C. These novel zymogen forms of protein C offer special advantages in the treatment of blood disorders involving coagulation.
  • Protein C a vitamin K dependent plasma protein, is of major physiological importance in the control of hemostasis and plays a significant role in the regulation of blood coagulation.
  • Protein C is synthesized as an inactive molecule, herein called nascent protein C. Nascent protein C undergoes complex processing, giving rise to a number of different inactive molecules as is more fully described below.
  • Inactive, secreted forms of protein C are referred to herein as zymogen protein C.
  • Activation of protein C occurs in the blood by a reaction involving a thrombomodulin-thrombin complex.
  • Activated protein C, together with its cofactor protein S is an anticoagulant of important physiological significance.
  • Activated protein C can prevent intravascular thrombosis and control the extension of existing clots.
  • the mechanism of action of the activated form of protein C and the mechanism of activation of the inactive zymogen into the active protease have been clarified in recent years (for review, see J. E. Gardiner and J. H. Griffin, Progress in Hematology, Vol. XIII, pp. 265-278, ed. Elmer B. Brown, Grune and Stratton, Inc., 1983).
  • thrombomodulin forms a tight, stoichiometric complex with thrombin.
  • thrombomodulin when complexed with thrombin, totally changes the functional properties of thrombin.
  • thrombomodulin normally clots fibrinogen, activates platelets, and converts clotting cofactors V and VIII to their activated forms, Va and Vllla.
  • thrombin activates protein C, but only very slowly and inefficiently, and the activation is further inhibited by physiological Ca2+.
  • thrombin complexed with thrombomodulin does not clot fibrinogen, activate platelets, or convert clotting factors V and VIII to their activated counterparts Va and Vllla, but does become a very efficient activator of protein C zymogen in the presence of physiological Ca2+.
  • the rate constant of protein C zymogen activation by thrombomodulin-thrombin is over 1,000 fold higher than the rate constant for thrombin alone.
  • the coagulation system is best looked at as a chain reaction involving the sequential activation of zymogens into active serine proteases. This chain reaction eventually produces the enzyme thrombin, which through limited proteolysis converts plasma fibrinogen into the insoluble gel fibrin.
  • Two key events in the coagulation cascade are the conversion of clotting factor X to Xa by clotting factor IXa and the conversion of prothrombin into thrombin by clotting factor Xa. Both of these reactions occur on cell surfaces, most notably the platelet surface, and both reactions require cofactors.
  • the major cofactors, factors V and VIII, in the system circulate as relatively inactive precursors, but when the first few molecules of thrombin are formed, thrombin loops back and activates the cofactors through limited proteolysis.
  • the activated cofactors, Va and Vllla accelerate both the conversion of prothrombin into thrombin and also the conversion of factor X to factor Xa by approximately five orders of magnitude.
  • Activated protein C preferentially acts on, to proteolytically degrade, hydrolyze, and irreversibly destroy clotting cofactors Va and Vllla, the activated forms of the inactive clotting factors V and VIII.
  • Clotting factors V and VIII in contrast, are very poor substrates for activated protein C in vivo.
  • Protein S an important cofactor for activated protein C is protein S, another vitamin K-dependent plasma protein. Protein S substantially increases activated protein C-mediated hydrolysis of factors Va and Villa 25 fold.
  • Protein C is recognized as a valuable therapeutic agent (see, for example, European Patent Publications Nos. 0215548 and 0191606).
  • Activated protein C is a novel antithrombotic agent with a wider therapeutic index than available anticoagulants, such as heparin and the oral hydroxycoumarin type anticoagulants.
  • zymogen protein C nor activated protein C is effective until thrombin is generated, because thrombin is needed to convert clotting factors V to Va and VIII to Vllla; the activated forms of these two cofactors are the preferred substrate for activated protein C.
  • Thrombin is also required to activate zymogen protein C, for without the thrombomodulin-thrombin complex, the protein C zymogen is not converted into its active counterpart.
  • Activated protein C is an on-demand anticoagulant, because activated protein C works by inactivating cofactors Va and Vllla. Because thrombin is required to convert factors V and VIII to their activated counterparts Va and Vllla, protein C only acts as an anticoagulant after thrombin is generated. Conventional anticoagulants, in contrast to activated protein C, maintain a constant anticoagulant state throughout the circulation for as long as they are given to the patient, thereby substantially increasing the risk of bleeding complications over that for protein C or activated protein C. Activated protein C is therefore an on-demand anticoagulant of wide clinical utility for use as an alternative to heparin and the hydroxycoumarins.
  • protein C zymogen is of great therapeutic importance.
  • congenital homozygous protein C deficiency affected individuals die in early childhood from purpura fulminans, an often lethal form of disseminated intravascular coagulation.
  • heterozygous protein C deficiency affected individuals suffer severe, recurrent thromboembolic episodes.
  • plasma protein concentrates designed to treat hemophilia B or factor IX deficiency which contain protein C as an impurity, are effective in the prevention and treatment of intravascular clotting in heterozygous protein C deficiency.
  • Protein C levels have also been noted to be abnormally low in thrombotic states such as disseminated intravascular coagulation and in disease states predisposing to thrombosis, such as major trauma, major surgery, and cancer.
  • amino acid residue sequence for nascent human protein C is depicted below. This amino acid residue sequence, and relevant portions thereof, also characterizes "native human protein C" for purposes of the present invention.
  • A is deoxyadenyl
  • G is deoxyguanyl
  • C is deoxycytidyl
  • T is thymidyl
  • ALA is Alanine
  • ARG is Arginine
  • ASN is Asparagine
  • ASP is Aspartic acid
  • -COOH is the carboxy terminus
  • CYS Cysteine
  • GLN is Glutamine
  • GLU Glutamic Acid
  • GLY Glycine
  • H 2 N is the amino terminus
  • HIS is Histidine
  • ILE is Isoleucine
  • LEU is Leucine
  • LYS is Lysine
  • MET is Methionine
  • PHE is Phenylalanine
  • PRO is Proline
  • SER Serine
  • THR Threonine
  • TRP is Tryptophan
  • TYR is Tyrosine
  • VAL Valine.
  • the DNA sequence depicted above was derived from cDNA clones prepared from human liver mRNA that encodes human protein C. Those skilled in the art recognize that the degenerate nature of the genetic code enables one to construct many different DNA sequences that encode the same amino acid residue sequence.
  • the cDNA sequence for nascent human protein C depicted above is thus only one of many possible nascent human protein C-encoding sequences.
  • a 5' poly G sequence, a 3' poly C sequence, and both 5' and 3' Pstl restriction enzyme recognition sequences were constructed at the ends of the protein C-encoding cDNA.
  • Plasmid pHC7 thus comprises the coding sequence above and, again depicting only one strand of the molecule, also contains these additional sequences: at the 5' and 3' ends, respectively, of the coding strand of the nascent human protein C coding sequence.
  • Plasmid pHC7 can be conventionally isolated from E. coli K12 RR1/pHC7, a strain deposited with and made part of the permanent stock culture collection of the Northern Regional Research Laboratory (NRRL), Peoria, Illinois.
  • NRRL Northern Regional Research Laboratory
  • a culture of E. coli K12 RR1/pHC7 can be obtained from the NRRL under the accession number NRRL B-15926.
  • a restriction site and function map of plasmid pHC7 is presented in Figure 2 of the accompanying drawings.
  • Nascent protein C can also be depicted schematically, as shown below.
  • Human protein C zymogen is a serine protease precursor synthesized in the liver and present in the blood. For expression of complete biological activity, protein C requires post-translational modifications for which vitamin K is needed.
  • the two-chain, disulfidelinked, protein C zymogen arises from the single-chain zymogen by limited proteolysis. This limited proteolysis is believed to include cleavage and removal of amino acid residues 198 and 199.
  • the activation of the two-chain zymogen into the active serine protease involves the proteolytic cleavage of an ARG-LEU peptide bond (residues 211 and 212).
  • Protein C is significantly glycosylated; the mature enzyme contains -23% carbohydrate. Protein C also contains a number of unusual amino acids, including y-carboxyglutamic acid and Q-hydroxyaspartic acid (erythro-L-,8-hydroxy aspartate).
  • y-carboxyglutamic acid (gla) is produced by y-glutamyl carboxylation from glutamic acid residues with the aid of a hepatic microsomal carboxylase which requires vitamin K as a cofactor.
  • human protein C can also be represented schematically and is shown below. Those skilled in the art recognize that the order of the steps shown in the schematic do not necessarily reflect the order of the steps in the in vivo pathway.
  • the present invention provides novel compounds, vectors, transformants, and methods for the recombinant expression of novel protein C zymogens.
  • Ad2LP the major late promoter of adenovirus type 2.
  • Figure 1 consists of four parts and schematically illustrates the construction protocol for plasmid pLPC, a starting material used in the construction of starting plasmid pLAPC.
  • Figure 1 Part A depicts the construction of plasmid pBKneo1 from BK virus and plasmid pdBPV-MMtneo.
  • Figure 1 Part B depicts the construction of plasmid pLPcat from adenovirus 2 and plasmid pSV2cat.
  • Figure 1 depicts the construction of plasmid pBLcat from plasmid pBKneo1 and plasmid pLPcat.
  • Figure 1 depicts the construction of plasmid pLPC from plasmid pBLcat and plasmid pL133.
  • Figure 2 schematically illustrates the construction of plasmid pL133, a starting material used in the construction of plasmid pLPC.
  • the present invention relates to DNA compounds that code for the expression of novel zymogen forms of human protein C.
  • Several methods of producing native human protein C zymogen and nascent human protein C have been described (see European Patent Publications 215548 and 191606). These prior art methods provide for the expression of zymogen forms of human protein C that do not differ from the zymogen forms present in human blood.
  • the protein C zymogen produced by these methods must be treated with substances such as a-thrombin, trypsin, or a mixture of thrombin and thrombomodulin (whether in vivo or in vitro) to obtain activated protein C.
  • a zymogen form of human protein C produced by recombinant DNA technology that is identical to zymogen forms of human protein C found naturally in human blood will only be activated in the body by the natural activation pathway involving the thrombin- thrombomodulin complex.
  • Native human protein C zymogen can be activated by thrombin alone; however, the activation requires the absence of Ca2+ and such high levels of thrombin and/or protein C zymogen that it is not a significant in vivo pathway to activated protein C.
  • the present invention provides zymogen forms of human protein C that can be activated in vivo by thrombin alone at a rate of clinical significance. In addition, these zymogen forms are much more susceptible to activation by thrombin/thrombomodulin than native human protein C zymogen.
  • the present invention also provides DNA compounds, recombinant DNA expression vectors, transformed cell lines, and methods for the recombinant expression of these novel zymogen forms of human protein C.
  • the method for producing these zymogen forms of human protein C comprises:
  • the invention also provides DNA compounds for use in the method of producing these novel zymogen forms of human protein C.
  • These novel compounds all encode a pre-propeptide comprising a signal peptide for directing secretion and a propeptide from a y-carboxylated (through the action of a vitamin K-dependent carboxylase) protein.
  • propeptide sequences are well-known in the art. See, for example, Suttie et al., 1987, Proc. Natl. Acad. Sci. 84:634-637.
  • both the signal peptide coding sequence and the propeptide coding sequence will be derived from the amino acid residue sequence of the pre-propeptide of a y-carboxylated protein.
  • y-carboxylatedproteins examples include, but are not limited to, factor VII, factor IX, factor X, prothrombin, protein S, protein Z, and, protein C.
  • a DNA sequence encoding the pre-propeptide of human protein C is most preferred for use in the vectors of the invention.
  • the DNA compounds of the invention further comprise the coding sequence for the light chain of human protein C positioned immediately adjacent to, downstream of, and in translational reading frame with the pre-propeptide coding sequence.
  • the light chain of human protein C contains amino acid residues 43 to 197, inclusive, of nascent protein C, as depicted in the background section above.
  • the amino-terminal portions of the vitamin K-dependent plasma proteins such as the amino-terminal portion of the light chain of protein C, are responsible for calcium-binding activity of these proteins.
  • the calcium-binding domains of these plasma proteins such as factor VII, factor IX, factor X, prothrombin, and protein S, are interchangeable (see European Patent Publication No. 0215548A1, at pages 12 and 13) and equivalent to the calcium-binding domain of the light chain of human protein C.
  • the DNA compounds of the invention further comprise the coding sequence for the dipeptide LYS-ARG (KR) positioned immediately adjacent to, downstream of, and in translational reading frame with the light chain coding sequence.
  • a dibasic dipeptide such as LYS-ARG is positioned in the nascent protein at the carboxyl-terminal side of the light chain. The orientation of the LYS-ARG dipeptide in the expressed protein is irrelevant for purposes of the present invention.
  • Dibasic dipeptides such as LYS-LYS or ARG-ARG are equivalent to the LYS-ARG dipeptide for purposes of the present invention.
  • the dipeptide LYS-ARG which is the dipeptide in native human protein C, is preferred.
  • the coding sequence of the activation peptide Immediately downstream of the codons for the LYS-ARG dipeptide is the coding sequence of the activation peptide.
  • changes in the activation peptide coding sequence are primarily responsible for the property of increased thrombin-sensitivity of these novel zymogens.
  • the zymogen forms of the present invention primarily differ from native zymogen forms of human protein C as described below.
  • native human protein C the activation peptide is: in which the numbers refer to the position of the amino acid residues in nascent human protein C.
  • the present invention discloses that changing the ASP residue at position 209 to either a PHE, GLY, TYR, or TRP residue will result in the corresponding zymogen form having a greater sensitivity to cleavage by thrombin alone, in addition to a greater sensitivity to cleavage by the thrombin-thrombomodulin complex.
  • resulting zymogen is used to indicate that although substitutions are described with reference to amino acid positions in nascent human protein C, nascent human protein C must first be secreted (resulting in removal of amino acid residues 1 through 42) to obtain a zymogen form. Substitution of the proline residue (in the activation peptide) at position 210 in nascent human protein C, in addition to one of the four substitutions at position 209 described above, for a valine residue thus results in a novel zymogen of the present invention.
  • the preferred novel zymogen forms of human protein C of the present invention result from secretion and processing of nascent human protein C molecules with the amino acid residue sequence depicted below: wherein R 1 is PHE, GLY, TYR, or TRP; R 2 is PRO or VAL; and R 3 is ASP or ASN.
  • the novel coding sequences of the invention can be readily constructed starting from a coding sequence for nascent human protein C from which the AP-encoding region has been deleted by site-specific mutagenesis. Shown schematically, this coding sequence has the structure: As described in the accompanying examples, this coding sequence was inserted into a recombinant DNA expression vector and the resulting vector was designated plasmid pLAPC. Plasmid pLAPC serves as useful starting material for the construction of illustrative vectors of the invention that drive high-level recombinant expression of the novel zymogen forms of human protein C of the invention. The construction protocol for plasmid pLAPC from starting plasmid pHC7 is described in Example 1. Plasmid pHC7 is available from the Northern Regional Research Center (NRRL), Peoria, IL 61604 in E. coli K12 RR1/pHC7 under the accession number NRRL B-15926.
  • NRRL Northern Regional Research Center
  • Plasmid pLPC-167G is an illustrative expression vector of the invention in which the codon for aspartic acid at position 209 in nascent human protein C has been changed to a codon for glycine.
  • the construction protocol for plasmid pLPC-167G is described in detail in Example 3. Essentially, the construction involved site-specific mutagenesis of the protein C coding sequence. A portion of the protein C coding sequence comprising the activation peptide-encoding DNA was isolated from plasmid pHC7, inserted into phage M13mp18, and then altered by site-specific mutagenesis.
  • the mutagenized coding sequence was then cloned into a eukaryotic cloning vector to achieve a plasmid, designated pLPC-167G, identical to plasmid pLAPC, except for the insertion of a coding sequence for the activation peptide in which the codon for glycine has been substituted for the codon for aspartic acid at position 209.
  • Plasmid pLPC-167F is an illustrative expression vector of the invention in which the codon for aspartic acid at position 209 in nascent human protein C has been changed to a codon for phenylalanine.
  • the construction protocol for plasmid pLPC-167F is described in detail in Example 4.
  • the construction protocol for plasmid pLPC-167F was substantially the same as the construction protocol for plasmid pLPC-167G.
  • these other compounds of the invention include the nascent proteins produced upon translation of the mRNA transcripts generated from the DNA coding sequences of the invention.
  • the compounds of the invention also include the zymogen forms generated upon secretion of the nascent proteins of the invention.
  • the activated protein C derivative produced upon activation of the zymogen form is also a compound of the invention.
  • the compounds of the invention include DNA coding sequences, expression vectors that drive expression of those sequences, nascent proteins produced upon translation of mRNA transcripts generated from those coding sequences, zymogens produced upon secretion of those nascent proteins, and activated derivatives of certain of the zymogens.
  • the coding sequence encodes an amino acid residue sequence identical to that of nascent human protein C except for the substitutions at positions 209, 210, and 214. These substitutions are depicted below in Table I.
  • DNA compounds of the invention can also be synthesized chemically, or by combining restriction fragments, or by a combination of techniques known in the art. DNA synthesizing machines are also available and can be used to construct the compounds of the invention.
  • the illustrative vectors of the invention comprise the BK enhancer positioned to stimulate transcription by the adenovirus major late promoter of the coding sequence of the invention.
  • a great number of eukaryotic promoters, enhancers, and expression vectors are known in the art and can be used in the method of the present invention.
  • a eukaryotic expression vector can function without an enhancer element.
  • the key aspect of the present invention does not reside in the particular enhancer, if any, or promoter, used to drive expression of the protein C zymogen but rather resides in the novel coding sequence and corresponding proteins produced from that sequence.
  • European Publication No. 0245949 discloses a number of expression vectors for native zymogen protein C that utilize the BK enhancer to stimulate a eukaryotic promoter positioned to drive expression of nascent human protein C. These vectors drive especially high expression levels when transformed into eukaryotic cells that also express an immediate-early gene product of a large DNA virus, such as the E1A gene product of adenovirus. As is evident from the illustrative vectors pLPC-167G and pLPC-167F disclosed herein, the BK enhancer-E1A gene product expression method is especially preferred for use with the vectors of the present invention.
  • the present invention is not limited to use in a particular eukaryotic host cell.
  • a variety of eukaryotic host cells are available from depositories such as the American Type Culture Collection (ATCC) Rockville, MD 20852, and are suitable for use with the vectors of the invention.
  • ATCC American Type Culture Collection
  • the choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of the protein C-encoding DNA compounds of the invention. Because nascent human protein C and the nascent human protein C derivatives of the invention undergo substantial post-translational modification, however, some host cells are more preferred for use with the vectors of the invention.
  • adenovirus-transformed, human embryonic kidney cells are especially preferred for use in the recombinant production of y-carboxylated proteins such as human protein C.
  • adenovirus-transformed, human embryonic kidney cell line is the 293 cell line, available from the ATCC under the accession number ATCC CRL 1573. The 293 cell line is also preferred for use with the vectors of the present invention.
  • adenovirus-transformed cell line in general are exceptional hosts for the production of y-carboxylated human protein C.
  • AV12 AV12-664
  • the AV12 cell line was constructed by injecting a Syrian hamster in the scruff of the neck with human adenovirus 12 and isolating cells from the resulting tumor.
  • the vectors of the invention can be transformed into and expressed in a variety of eukaryotic, especially mammalian, host cells.
  • Vectors of the invention that possess no selectable marker with which to isolate and identify stable eukaryotic transformants are useful not only for purposes of transient assay but also for purposes of cotransformation, a procedure disclosed in U.S. Patent No. 4,399,216.
  • the vectors of the invention can also comprise sequences that allow for replication in E. coli, as it is usually more efficient to prepare plasmid DNA in E. coli than in other host organisms.
  • Expression of the coding sequences for human protein C contained on the vectors of the invention occurs in those host cells in which the particular promoter associated with the structural gene functions. Exemplary host cells suitable for use in the invention are listed in Table II, along with appropriate comments.
  • the pSV2-type vectors comprise segments of the SV40 genome that constitute a defined eukaryotic transcription unit--promoter (ep), intervening sequence (IVS), and polyadenylation (pA) site.
  • ep eukaryotic transcription unit--promoter
  • IVS intervening sequence
  • pA polyadenylation
  • the plasmid pSV2-type vectors transform mammalian and other eukaryotic host cells by integrating into the host cell chromosomal DNA.
  • Plasmid pSV2-type vectors have been constructed (see Eukaryotic Viral Vectors, edited by Gluzman, published by Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1982), such as plasmids pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-hyg, and pSV2-,8-globin, in which the SV40 promoter drives transcription of an inserted gene.
  • These vectors are suitable for use with the coding sequences of the invention and are available either from the American Type Culture Collection (ATCC) in Rockville, Maryland or from the Northern Regional Research Laboratory (NRRL) in Peoria, Illinois.
  • ATCC American Type Culture Collection
  • NRRL Northern Regional Research Laboratory
  • Plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolate reductase (dhfr) gene under the control of the SV40 early promoter. Under the appropriate conditions, the dhfr gene is known to be amplified, or copied, in the host chromosome. This amplification, described in a review article by Schimke, 1984, Cell 37:705-713, can involve DNA sequences closely contiguous with the dhfr gene, such as a nascent human protein C-encoding sequence of the invention, and thus can be used to increase production of the protein C zymogens of the invention.
  • dhfr murine dihydrofolate reductase
  • Plasmids which were constructed for expression of the nascent protein C and protein C zymogens of the invention in mammalian and other eukaryotic host cells can utilize a wide variety of promoters.
  • the present invention is in no way limited to the use of the particular eukaryotic promoters exemplified herein. Promoters such as the SV40 late promoter or the eukaryotic promoters disclosed in Bucher et al., 1986, Nuc. Acids Res.
  • promoters from eukaryotic genes can be readily isolated and modified for use on recombinant DNA expression vectors designed to produce human protein C zymogen in eukaryotic host cells.
  • Eukaryotic promoters can also be used in tandem to drive expression of a coding sequence of the invention.
  • retroviruses are known that infect a wide range of eukaryotic host cells. The long terminal repeats in the retrovirus DNA often encode promoter activity and thus can be used to drive expression of the coding sequences of the invention.
  • Plasmid pRSVcat (ATCC 37152) comprises portions of the long terminal repeat of the Rous Sarcoma virus (RSV), a virus known to infect chicken and other host cells.
  • the RSV long terminal repeat sequences can be isolated on an -0.76 kb Ndel-Hindlll restriction fragment of plasmid pRSVcat.
  • the promoter in the RSV long terminal repeat (Gorman et al., 1982, P.N.A.S. 79:6777) is suitable for use in vectors of the invention.
  • Plasmid pMSVi (NRRL B-15929) comprises the long terminal repeats of the Murine Sarcoma virus (MSV), a virus known to infect mouse and other host cells.
  • the mouse metallothionein (MMT) promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the vectors of the invention.
  • the MMT promoter is present in the 15 kb plasmid pdBPV-MMTneo (ATCC 37224), which can serve as the starting material for the construction of other plasmids of the present invention.
  • the degeneracy of the genetic code allows for the substitution of nucleotides throughout polypeptide coding regions, as well as in the translational stop signal, without alteration of the encoded polypeptide coding sequence.
  • substitutable sequences can be deduced from the known amino acid or DNA sequence of human protein C and can be constructed by following conventional synthetic or site specific mutagenesis procedures. Synthetic methods can be carried out in substantial accordance with the procedures of Itakura et al., 1977 Science 198:1056 and Crea et al., 1978, Proc. Nat. Acad. Sci. USA 75:5765. Therefore, the present invention is in no way limited to the DNA sequences and plasmids specifically exemplified.
  • transformants After transformation of a vector of the invention into a eukaryotic host cell, one can select transformants on the basis of a selectable phenotype.
  • This selectable phenotype can be conferred either by a selectable marker present on the expression vector or present on another vector cotransformed with the expression vector into the host cell.
  • transformants it is desirable to identify which transformants are expressing the highest levels of the desired protein encoded on the expression vector. Such identification is especially important after a cotransformation procedure, which generates a number of transformants that contain only the plasmid containing the selectable marker and so do not contain the expression vector.
  • Example 6 a protocol not only for identifying cells that express and secrete a desired protein but also for quantifying, relative to the other cells examined using the method, the amount of protein secreted is described.
  • the protocol also allows for the isolation of viable cells secreting the highest levels of a desired protein.
  • Activated protein C has substantial antithrombotic properties in the prevention of extension of intravenous thrombi, in the prevention of formation of arterial thrombi, and in the prevention of death and organ failure from Gram negative sepsis, endotoxemia, and disseminated intravascular coagulation.
  • infusion of native zymogen protein C was without effect in the treatment of Gram negative septicemia with shock and disseminated intravascular coagulation (DIC).
  • Tt short half-life
  • Activated protein C complexes with a newly described APC inhibitor as well as with alpha-2 macroglobulin.
  • the inactive zymogens including the protein C zymogens of the invention, do not react with serine protease inhibitors.
  • the advantage of the protein C zymogens of this invention is that they are better activated by thrombin than native protein C zymogen, because thrombin no longer has an absolute requirement for complexing with thrombomodulin to activate these zymogens in the presence of Ca2+. It follows that these protein C zymogens, when administered, can be activated at sites of intravascular thrombin generation, i.e., at any site where an intravascular thrombus is under developement. Thus, these recombinant protein C zymogens can be used as pro drugs and will become activated only at the sites of thrombin generation. Because these thrombin-sensitive zymogens can be administered in the zymogen form, they will not complex with protein C inhibitors and will exhibit a biological half-life equal to that of native protein C zymogen.
  • the recombinant protein C zymogens of the invention are useful in the prevention and treatment of a wide variety of acquired disease states involving intravascular coagulation, including deep vein thrombosis, pulmonary embolism, peripheral arterial thrombosis, emboli originating from the heart or peripheral arteries, acute myocardial infarction, thrombotic strokes, and dis-seminated intravascular coagulation.
  • These protein C derivatives can also be used efficiently in the treatment of the significant numbers of patients with heterozygous protein C deficiencies presenting recurrent deep vein thrombosis and in the case of the homozygous protein C deficient patients with purpura fulminans.
  • the doses of the protein C zymogens of the invention can be substantially reduced in clinical situations, as compared to activated protein C.
  • the dose of a protein C zymogen of the invention will range from about 5 mg to 100 mg per treatment, and in heterozygous protein C deficiency, the dose will range from about 2.5 mg to 50 mg per treatment.
  • activated protein C An attractive therapeutic indication for activated protein C is in the prevention of deep vein thrombosis and pulmonary embolism, currently treated with low doses of heparin.
  • the dose of recombinant activated protein C for prevention of deep vein thrombosis is in the range from 1-10 mg/day.
  • the dose of a protein C zymogen of the invention will range from about 0.25 to 5 mg per day. The added advantage of these zymogens is that they may be given as bolus injections rather than constant IV infusions. Activated protein C must be given by continuous IV infusion because of the short T1 ⁇ 2 of that protein.
  • the dose of activated protein C ranges from 1 - 10 mg as a loading dose followed by a continuous infusion in amounts ranging from 3-30 mg/day.
  • the protein C zymogens of the invention may be given by repeated bolus injection in doses not to exceed about 12 mg per 24 hours.
  • the protein C zymogens of the invention can be used for the treatment of emboli originating from thrombi in peripheral arteries, most notably the carotid arteries, which are not treated or prevented satisfactorily with currently used regimens, which include drugs capable of suppressing platelet function, oral anticoagulants, or combinations thereof.
  • these zymogens can be administrated long term in the same manner as outlined for cardiogenic emboli and have major potential in the prevention of emboli originating from carotid artery thrombi and resulting in embolic strokes.
  • the protein C zymogens of the invention are also useful in thrombotic strokes.
  • strokes are not usually treated with conventional anticoagulants.
  • Treatment of strokes with either heparin or oral anticoagulants, although occasionally beneficial, carries a high risk for bleeding into the infarcted brain area, thereby aggravating the neurological deficit accompanying the stroke.
  • the zymogens of the invention can be given to stroke victims and can be beneficial in preventing the local extension of the occluding arterial thrombus, thereby reducing the neurological deficit resulting from the stroke.
  • the amount of the zymogen effective in the treatment of stroke will be lower, as compared with activated protein C, but the dose will vary with each patient depending on the nature and severity of the stroke.
  • the zymogens of the invention will also be useful in treating acute myocardial infarction, because of their pro-fibrinolytic properties, once activated. These zymogens can be given with tissue plasminogen activator during the acute phases of the myocardial infarction. After the occluding coronary thrombus is dissolved, the zymogens can be given for additional days to prevent acute myocardial reinfarction. If activated protein C is administered in this situation, the patient is given a loading dose of 1-10 mg at the time plasminogen activator treatment is initiated followed by a continuous infusion of activated protein C ranging from 3-30 mg/day. In contrast, the zymogens of the invention can be given through bolus injection 3 to 4 times a day in doses not to exceed about 12 mg/day.
  • Activated protein C is useful in the treatment of disseminated intravascular coagulation.
  • Heparin and the oral anticoagulants have been given to patients with disseminated intravascular coagulation (DIC) in extensive clinical trials, but the results have been disappointing.
  • DIC disseminated intravascular coagulation
  • activated protein C, as well as the zymogens of the present invention has a distinct advantage over conventional anticoagulants. As mentioned above, it has been established in animal experiments that the protein C zymogen is ineffective in the prevention of death and organ damage from Gram negative septicemia and disseminated intravascular coagulation.
  • the protein C zymogens of the invention being highly susceptible to activation by thrombin, will be effective treatment for disseminated intravascular coagulation.
  • the estimated requirements for activated protein C to treat DIC is approximately 100 mg/day; the doses of the zymogen forms of the invention for treatment of DIC are not to exceed about 30 mg/day, administered as repeated bolus injections.
  • Conventional anticoagulant drugs are useful in the treatment of invasive malignant tumors.
  • Many tumor cells produce substances which trigger the activation of the coagulation system resulting in local fibrin deposits. These fibrin deposits function as "nests" in which cancer cells can divide to form metastatic lesions.
  • warfarin or other conventional anticoagulants in combination with the more intensive and effective forms of chemotherapy, because such therapy always produces a sharp drop in the platelet count, and thrombocytopenia combined with warfarin therapy puts the patient at an unacceptably high risk for serious bleeding complications.
  • the protein C derivatives of the invention like activated protein C, being more selective than conventional anticoagulants and having a far higher therapeutic index than either heparin or the oral anticoagulants, can be given relatively safely to the thrombocytopenic patient, thus making possible the treatment of patients with invasive cancers with effective and intensive chemotherapy in combination with a protein C zymogen of the invention. Treatment will follow a dosage regimen comparable to that used in deep vein thrombosis- pulmonary embolism.
  • the zymogens, and activated counterparts, of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby a human protein C zymogen or activated protein C of the invention is combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • a pharmaceutically acceptable carrier vehicle e.g., human serum albumin.
  • Such compositions will contain an effective amount of a protein C zymogen, or activated counterpart, together with a suitable amount of carrier vehicle to prepare pharmaceutically acceptable compositions suitable for effective administration to the host.
  • the protein C composition can be administered parenterally, or by other methods that ensure its delivery to the bloodstream in an effective form.
  • the zymogens of the present invention can be used to prepare activated protein C in vitro. Although recombinant methods for producing activated protein C directly in eukaryotic cells are known, these methods require that the activated protein C remain in the culture media for long periods of time. In addition, the activated protein C must be purified from the culture medium, an expensive, multi-step process. Because activated protein C is relatively unstable, these direct expression methods can yield low amounts of activated protein C. In contrast, the zymogens of the invention can be activated by thrombin alone, even in the presence of Ca2+, and thus offer significant advantages over known methods for producing activated protein C.
  • Example 1A describes the isolation of a DNA fragment encoding a portion of the protein C molecule, including the activation peptide, from plasmid pHC7.
  • Example 1 B describes the cloning of this DNA fragment into phage M13mp18 and the removal of the DNA encoding the activation peptide from the resulting recombinant phage by site specific mutagenesis.
  • Example 1 C describes the final steps in the construction of plasmid pLAPC, more specifically, the isolation of the mutagenized fragment and its ligation with two fragments derived from plasmid pLPC to yield plasmid pLAPC.
  • the construction protocol for plasmid pLPC is described in Example 2.
  • Plasmid pHC7 contains the complete coding sequence for nascent human protein C.
  • L broth (10 g peptone, 10 g NaCI, and 5 g yeast extract) containing 15 ⁇ g/ml tetracycline was inoculated with a culture of E. coli K12 RR1/pHC7 (NRRL B-15926) and incubated in an air-shaker incubator at 37°C until the optical density (O.D.) at 590 nm was -1 absorbance unit, at which time 150 mg of chloramphenicol were added to the culture. The incubation was continued for about 16 hours; the chloramphenicol addition inhibits protein synthesis, and thus inhibits further cell division, but allows plasmid replication to continue.
  • O.D. optical density
  • the lysed cells were frozen in a dry ice-ethanol bath and then thawed.
  • the cellular debris was removed from the solution by centrifugation at 25,000 rpm for 40 minutes in an SW27 rotor (Beckman, 7360 N. Lincoln Ave., Lincolnwood, IL 60646). About 30.44 g of CsCI and -1 ml of a 5 mg/ml ethidium bromide solution were added to the solution, the volume of which was then adjusted to 40 ml. The solution was decanted into a Vti50 ultra-centrifuge tube (Beckman). The tube was sealed and then centrifuged in a Vti50 rotor at 42,000 rpm for -16 hours.
  • the resulting plasmid band visualized with ultraviolet light, was isolated and then placed in a ti75 tube and rotor (Beckman) and centrifuged at 55,000 rpm for 16 hours. Any necessary volume adjustments were made using TES containing 0.761 g/ml CsCI.
  • the plasmid band was again isolated, the ethidium bromide extracted with salt-saturated isopropanol, and finally diluted 1:3 with TES buffer. Two volumes of ethanol were then added to the solution, and the resulting mixture was incubated overnight at -20 ° C.
  • the plasmid DNA was pelleted by centrifuging the solution in an SS34 rotor (DuPont Co.) for 15 minutes at 10,000 rpm.
  • the reaction mixture was then electrophoresed on an -0.6% low-gelling-temperature agarose (FMC Corporation, Marine Colloids Division, Rockland, Maine 04841) gel for 2-3 hours at -130 V and -65 mA in Tris-Acetate buffer.
  • the gel was stained in a dilute solution of ethidium bromide, and the band of DNA constituting the -0.7 kb Sstl-Sall restriction fragment, which was visualized with long-wave UV light, was cut from the gel in a small segment.
  • the volume of the segment was determined by weight and density of the segment, and four volumes of TE containing 0.25 M NaCl were added to the tube containing the segment. The segment was then melted by incubation at 72 °C.
  • phage M13mp18 obtained from New England Biolabs
  • RF replicaative form
  • DNA was digested with restriction enzymes Sstl and Sail in substantial accordance with the procedure described in Example 1A.
  • the reaction was stopped by extracting the reaction mixture with phenol and then chloroform; then, the DNA was precipitated, collected by centrifugation, and resuspended in about 15 ⁇ l of TE buffer.
  • the two fragments resulting from the digestion were separated on an -0.6% low-gelling-temperature agarose gel, and the larger fragment was cut out from the gel and purified as described in Example 1A.
  • E. coli K12 JM101 (New England Biolabs) were used to inoculate 30 ml of 2X TY broth (TY broth is 10 g/L tryptone, 10 g/L NaCI, and 5 g/L yeast extract), and the resulting culture was incubated at 37 ° C with aeration until the O.D. 6 oo was -0.5.
  • the culture was chilled for 10 minutes in an ice-water bath, collected by centrifugation, and resuspended in 15 ml of cold, 10 mM NaCI.
  • the cells were again collected by centrifugation and then resuspended in 15 ml of cold, 30 mM CaC1 2 .
  • the cells were placed on ice for 20 minutes and collected by centrifugation. The cells were resuspended in 1.5 ml of cold, 30 mM CaCI 2 ; a 200 ⁇ l aliquot of the cells was removed, added to 9 ⁇ l of the ligated DNA prepared above, and incubated on ice for about 30 minutes.
  • the cell-DNA mixture was then incubated at 42 ° C for 2 minutes and then added to 3 ml of top agar (TY broth with 0.5% agar kept molten at 45 ° C) that also contained 50 ⁇ l of 2% X-Gal ("X-Gal” is 5-Bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside), 50 ⁇ l of 100 mM IPTG ("IPTG” is isopropyl ⁇ -D-thiogalactopyranoside), and 100 ⁇ l of E. coli K12 JM101 in logarithmic growth phase. The cell-top agar mixture was then plated on TY-agar plates, and the plates were incubated at 37 ° C overnight.
  • X-Gal is 5-Bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside
  • IPTG is isopropyl ⁇ -D-thiogalactopyranoside
  • the phage RF DNA was isolated from the cell pellets using a scaled-down version of the procedure described in Example 1A, except that no antibiotic was used in the culture media, and the ultracentrifugation steps were replaced with phenol and chloroform extractions.
  • Transformants containing phage M13mp18-HE1 DNA were identified by restriction enzyme analysis of their phage DNA.
  • the overnight cultures were centrifuged, and about 1 ml of a solution composed of 20% polyethylene glycol (PEG) 6000 and 2.5 mM NaCl was added per 5 ml of supernatant, which was then incubated at room temperature for 10 minutes.
  • the DNA solution was extracted first with chloroform, then twice with TE-saturated phenol, and then again with chloroform.
  • the single-stranded DNA was then precipitated using NaOAc and ethanol, centrifuged, and, after the pellet was washed with 70% ethanol and dried, the resulting pellet was dissolved in 80 ⁇ l of H 2 0. This phage preparation was used in the next step, the site-specific mutagenesis, to remove the activation peptide-encoding DNA.
  • the single-stranded DNA fragment used in the mutagenesis to remove the activation peptide-encoding DNA was synthesized on an automated DNA synthesizer and is depicted below:
  • the mutagenic oligonucleotide and the M13 universal primer were annealed to the single-stranded phage DNA.
  • the phage DNA was made double-stranded by extending the primers with DNA polymerase.
  • the extension reaction was incubated at room temperature for 30 minutes, then at 37 ° C for 4 hours, and then overnight at 4 ° C.
  • the reaction was stopped by a phenol-chloroform extraction and precipitation of the DNA with ethanol and sodium acetate (NaOAc).
  • the solution of DNA was split equally into two tubes, and to one of the tubes, 100 units (BMB) of S1 nuclease were added.
  • BMB 100 units
  • the S1 reaction was incubated at room temperature for 5 minutes and stopped by extracting the reaction mixture once with TE-saturated phenol-chloroform (50:50).
  • the DNA was precipitated from the reaction mixture and from the non-S1-treated sample with NaOAc and ethanol.
  • the DNA pellets were resuspended in 60 ⁇ l of H 2 0 and used to transform E. coli K12 JM101 in accordance with the procedure used during the construction of phage M13mp18-HE1, except that no IPTG or X-Gal was added to the plates.
  • the mutants were screened for by using a small portion of the mutagenic oligonucleotide, 5'-TGAAACGACTCATTGA-3' (radioactively labelled), as a probe in plaque and dot-blot hybridizations.
  • 5'-TGAAACGACTCATTGA-3' radioactively labelled
  • the single-stranded DNA was sequenced using the dideoxy-sequencing method (J.H. Smith, 1980, Methods in Enzymology 65:560-580).
  • Several phage were identified with the desired mutation. Phage in which the coding sequence for the activation peptide was deleted were designated phage M13mp18-HE2.
  • the mutation in phage M13mp18-HE2 causes a decrease in size of 36 bp with respect to the natural coding sequence, a difference that can be used to facilitate identification of DNA that contains the mutated region.
  • the RF form of phage M13mp18-HE2 was prepared for use in subsequent constructions.
  • the mutagenized Sstl-Sall (-0.7 kb) restriction fragment of the RF form of phage M13mp18-HE2 was cut from the phage and isolated in substantial accordance with the procedure of Example 1A. However, the -100 ⁇ l of solution containing -0.1 ⁇ g of the desired 0.7 kb fragment in a 1:2 dilution of low-gelling agarose were not passed through any purification column but were used directly in the ligation to produce plasmid pLAPC, described below.
  • Plasmid pLAPC Three DNA fragments were ligated together to form plasmid pLAPC: the -0.7 kb Sstl-Sall restriction fragment of phage M13mp18-HE2, described above, and two DNA fragments from plasmid pLPC.
  • the construction protocol for plasmid pLPC is described in Example 2.
  • a restriction site and function map of plasmid pLPC is presented in Figure 1 of the accompanying drawings. Because of the positioning of Sall, Sstl, and EcoRl restriction enzyme recognition sites on plasmid pLPC, the desired EcoRI-Sall and EcoRl-Sstl restriction fragments had to be prepared in two separate digestions.
  • the ligated DNA containing plasmid pLAPC was used to transform E. coli K12 RV308, available from the NRRL under the accession number NRRL B-15624.
  • a 50 ml culture of E. coli K12 RV308 in L broth was grown to an optical density (O.D.) at 590 nm of -0.6.
  • the culture was chilled on ice for ten minutes, and the cells were collected by centrifugation.
  • the cell pellet was resuspended in 25 ml of cold, 10 mM NaCI.
  • the cells were again pelleted by centrifugation, and the pellet was resuspended in 25 ml of cold, 30 mM CaC1 2 and incubated on ice for 30 minutes.
  • the cells were again collected by centrifugation and resuspended in 2.5 ml of cold, 30 mM CaCI 2 .
  • E. coli K12 RV308/pLAPC transformants were verified by restriction enzyme analysis of their plasmid DNA. Plasmid DNA was obtained from the E. coli K12 RV308/pLAPC transformants in substantial accordance with the teaching of Example 1A, except that 50 ⁇ g/ml of ampicillin, and not tetracycline, was used as the selective agent.
  • Plasmid pLPC was used as an intermediate vector in the construction of plasmid pLAPC (see Example 1 C). Plasmid pLPC comprises a segment of DNA that encodes the BK virus enhancer and the adenovirus 2 late promoter positioned to drive expression of human protein C.
  • the construction protocol for plasmid pLAPC essentially results in the replacement of the human protein C coding sequence on plasmid pLPC with another protein C coding sequence from which the activation peptide-encoding DNA has been removed.
  • the BK enhancer/adenovirus late promoter expression control sequences on plasmids pLPC and pLAPC are greatly stimulated in activity by the presence of an immediate early gene product of a large DNA virus, i.e., the E1A gene product of adenovirus.
  • Example 2A describes the isolation of BK virus DNA, from which the BK enhancer can be obtained.
  • Example 2B sets forth the construction protocol for plasmid pBKneol, a plasmid resulting from the insertion of the BK enhancer into plasmid pdBPV-MMTneo.
  • Example 2C teaches the construction protocol for plasmid pLPcat, a plasmid resulting from the insertion of the adenovirus 2 late promoter into plasmid pSV2cat.
  • Example 2D teaches the construction protocol for plasmid pBLcat, a plasmid that contains the BK enhancer positioned to stimulate the activity of the adenovirus late promoter.
  • Example 2E describes the construction protocol for plasmid pL133, a protein C expression vector, beginning with starting material plasmid pHC7 and proceeding through the construction of intermediate plasmid pSV2-HPC8 and then the final construction of plasmid pL133.
  • Example 2F teaches the construction protocol for plasmid pLPC, which comprises the BK enhancer/adenovirus late promoter expression control sequence of plasmid pBLcat inserted into plasmid pL133 to drive expression of human protein C.
  • BK virus is obtained from the American Type Culture Collection under the accession number ATCC VR-837. The virus is delivered in freeze-dried form and resuspended in Hank's balanced salts (Gibco, 3175 Staley Road, Grand Island, NY 14072) to a titer of about 10 5 plaque-forming units (pfu)/ml.
  • the host of choice for the preparation of BK virus DNA is primary human embryonic kidney (PHEK) cells, which can be obtained from Flow Laboratories, Inc., 7655 Old Springhouse Road, McLean, VA 22101, under catalogue number 0-100 or from M.A. Bioproducts under catalogue number 70-151.
  • PHEK primary human embryonic kidney
  • the virus is released from the cells by three freeze-thaw cycles, and the cellular debris is removed by centrifugation at 5000Xg.
  • the virus in 1 liter of supernatant fluid is precipitated and collected by the addition of 100 g of PEG-6000, incubation of the solution for 24 hours at 4 ° C, and centrifugation at 5000Xg for 20 minutes.
  • the virus suspension is layered onto a 15 ml solution of saturated KBr in a tube, which is centrifuged at 75,000Xg for 3 hours.
  • SDS Sodium dodecyl sulfate
  • pronase@ (Sigma) protease is added to a concentration of 100 ⁇ g/ml, and the solution is incubated at 37 ° C for 2 hours.
  • Cesium chloride is then added to the solution to a density of 1.56 g/ml, and ethidium bromide is added to the solution to a final concentration of 100 ⁇ g/ml.
  • the solution is centrifuged in a Sorvall 865 rotor (DuPont Co., Newton, CT 06470) or similar vertical rotor at 260,000Xg for 24 hours.
  • the solution of BK virus DNA is then dialyzed against TE buffer until the 260 nm/280 nm absorbance ratio of the DNA is between 1.75 and 1.90.
  • the DNA is precipitated by adjusting the NaCl concentration to 0.15 M, adding two volumes of ethanol, incubating the solution at -70 °C for at least 2 hours, and centrifuging the solution at 12,000Xg for 10 minutes.
  • the resulting pellet of BK virus DNA is suspended in TE buffer at a concentration of 1 mg/ml.
  • a restriction site and function map of BK virus is presented in Figure 1 of the accompanying drawings.
  • E. coli K12 HB101/pdBPV-MMTneo cells are obtained in lyophilized form from the American Type Culture Collection under the accession number ATCC 37224.
  • the lyophilized cells are plated on L-agar plates containing 100 ⁇ g/ml ampicillin and incubated at 37°C to obtain single colony isolates.
  • the -1 mg of plasmid pdBPV-MMTneo DNA obtained by this procedure was suspended in 1 ml of TE buffer and stored at -20 °C.
  • the plasmid isolation procedure described in Example 1A is generally used when large amounts of very pure plasmid DNA are desired.
  • the procedure can be modified to obtain rapidly a smaller, less pure amount of DNA, such as is needed when screening transformants for the presence of a given plasmid, by using only about 5 ml of cultured cells, lysing the cells in an appropriately scaled-down amount of lysis buffer, and replacing the centrifugation steps with phenol and chloroform extractions.
  • E. coli K12 HB101 cells are available in lyophilized form from the Northern Regional Research Laboratory under the accession number NRRL B-15626.
  • a 50 ml culture of E. coli K12 HB101 in L broth was grown to an optical density at 650 nanometers (O.D. 650 ) of approximately 0.4 absorbance units.
  • the culture was chilled on ice for ten minutes, and the cells were collected by centrifugation.
  • the cell pellet was resuspended in 25 ml of cold 100 mM MgC1 2 and incubated on ice for 25 minutes.
  • the cells were once again pelleted by centrifugation, and the pellet was resuspended in 2.5 ml of cold 100 mM CaC1 2 and incubated for 30 minutes on ice. After the incubation, the cells are competent for the uptake of transforming DNA.
  • the virion DNA of adenovirus 2 is a double-stranded linear molecule about 35.94 kb in size.
  • the Ad2 late promoter can be isolated on an -0.32 kb Accl-Pvull restriction fragment of the Ad2 genome; this -0.32 kb restriction fragment corresponds to the sequence between nucleotide positions 5755 and 6071 of the Ad2 genome.
  • Ad2 DNA is first digested with restriction enzyme Ball, and the -2.4 kb Ball restriction fragment that comprises the entire sequence of the -0.32 kb Accl-Pvull restriction fragment is isolated. Then, the -2.4 kb Ball restriction fragment is digested with Accl and Pvull to obtain the desired fragment.
  • the Ball-digested DNA is loaded onto an agarose gel and electrophoresed until the restriction fragments are well separated. Visualization of the electrophoresed DNA is accomplished by staining the gel in a dilute solution (0.5 ⁇ g/ml) of ethidium bromide and exposing the stained gel to long-wave ultraviolet (UV) light.
  • a dilute solution 0.5 ⁇ g/ml
  • UV long-wave ultraviolet
  • One method to isolate DNA from agarose is as follows. A small slit is made in the gel in front of the desired fragment, and a small piece of NA-45 DEAE membrane (Schleicher and Schuell, Keene, NH 03431) is placed in each slit. Upon further electrophoresis, the DNA non-covalently binds to the DEAE membrane.
  • the volume of the high salt-DNA solution is adjusted so that the NaCl concentration is 0.25 M, and then three volumes of cold, absolute ethanol are added to the solution.
  • the resulting solution is mixed and placed at -70 °C for 10-20 minutes.
  • the solution is then centrifuged at 15,000 rpm for 15 minutes. After another precipitation to remove residual salt, the DNA pellet is rinsed with ethanol, dried, resuspended in 20 ⁇ l of TE buffer, and constitutes about 3 ⁇ g of the desired restriction fragment of Ad2.
  • the purified fragment obtained is dissolved in 10 ⁇ l of TE buffer.
  • the Accl-Pvull-digested, -2.4 kb Ball restriction fragment of Ad2 is loaded onto an -6% polyacrylamide gel and electrophoresed until the 0.32 kb Accl-Pvull restriction fragment that comprises the Ad2 late promoter is separated from the other digestion products.
  • the gel is stained with ethidium bromide and viewed using UV light, and the segment of gel containing the -0.32 kb Accl-Pvull restriction fragment is cut from the gel, crushed, and soaked overnight at room temperature in -250 ⁇ l of extraction buffer (500 mM NH 4 0Ac; 10 mM MgOAc; 1 mM EDTA; and 0.1% SDS).
  • the mixture is centrifuged, and the pellet is discarded.
  • the DNA in the supernatant is precipitated with ethanol; about 2 ⁇ g of tRNA are added to ensure complete precipitation of the desired fragment.
  • About 0.2 ⁇ g of the -0.32 kb Accl-Pvull restriction fragment are obtained and suspended in 7 ⁇ l of H 2 0.
  • Bcll linkers were ligated to the -0.32 Accl-Pvull restriction fragment. Because the Bcll linkers were blunt-ended, the linkers only attached to the Pvull end of the restriction fragment.
  • the Bcll linkers (New England Biolabs), which had the following sequence: were kinased and prepared for ligation by the following procedure.
  • E. coli K12 HB101/pSV2cat cells are obtained in lyophilized form from the ATCC under the accession number ATCC 37155, and plasmid pSV2cat DNA was isolated from the cells in substantial accordance with the procedure of Example 1A, except that ampicillin, at 50 ⁇ g/ml, was used in place of tetracycline.
  • a restriction site and function map of plasmid pSV2cat is presented in Figure 1, Part B, of the accompanying drawings. About 1 mg of plasmid pSV2cat DNA is obtained and dissolved in 1 ml of TE buffer.
  • ligation reaction was incubated at 16°C overnight.
  • the ligated DNA constituted the desired plasmid pLPcat, a plasmid that comprises the Ad2 late promoter positioned so as to drive transcription, and thus expression, of the chloramphenicol acetyltransferase gene.
  • a restriction site and function map of plasmid pLPcat is presented in Figure 1, Part B, of the accompanying drawings.
  • the ligated DNA was used to transform E. coli K12 HB101 cells in substantial accordance with the procedure of Example 2B.
  • the transformed cells were plated on L-agar plates containing 50 ⁇ g/ml ampicillin; restriction enzyme analysis of plasmid DNA was used to identify the E. coli K12 HB101/pLPcat transformants.
  • Plasmid pLPcat DNA was isolated from the transformants for use in subsequent constructions in substantial accordance with the plasmid isolation procedure described in Example 1A, except that ampicillin was used as the selective agent in place of tetracycline.
  • plasmid pBKneo1 DNA in 50 ⁇ l of TE buffer were added to 7.5 ⁇ l of 10X Accl buffer, 30 ⁇ l of H 2 0, and 15 ⁇ l (about 75 units) of restriction enzyme Accl, and the resulting reaction was incubated at 37 °C for 2 hours.
  • the Accl-digested plasmid pBKneol DNA was loaded on an agarose gel, and the -1.4 kb fragment that contains the BK enhancer was separated from the other digestion products. The -1.4 kb Accl restriction fragment was then isolated from the gel and purified.
  • plasmid pLPcat DNA was dissolved in 5 ⁇ l of 10X Accl buffer and 40 ⁇ l of H 2 O. About 5 ⁇ l (-25 units) of restriction enzyme Accl were added to the solution of plasmid pLPcat DNA, and the resulting reaction was incubated at 37 °C. The Accl-digested plasmid pLPcat DNA was precipitated with ethanol and resuspended in 5 ⁇ l of 10X Stul buffer, 40 ⁇ l of H 2 0, and 5 ⁇ l (about 25 units) of restriction enzyme Stul, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Accl-Stul-digested plasmid pLPcat DNA was precipitated with ethanol several times to purify the -4.81 kb Accl-Stul restriction fragment that comprises the E. coli origin of replication and Ad2 late promoter away from the other digestion product, a restriction fragment about 16 bp in size. About 1 ⁇ g of the desired -4.81 kb restriction fragment was obtained and dissolved in 20 ⁇ l of TE buffer.
  • E. coli K12 HB101 cells were used to transform E. coli K12 HB101 cells in substantial accordance with the procedure described in Example 2B.
  • E. coli K12 HB101/pBLcat transformants were identified by restriction enzyme analysis of their plasmid DNA.
  • Plasmid pBLcat DNA was prepared for use in subsequent constructions in substantial accordance with the procedure of Example 1A, except that ampicillin was used as the selective agent in place of tetracycline.
  • Plasmid pL133 is a human protein C expression vector. As described below, plasmid pL133 can be constructed using starting vector plasmids pSV2gpt and pHC7 (the preparation of plasmid pHC7 is described above in Example 1A) to construct intermediate vector plasmid pSV2-HPCB, which is then combined with plasmid pSV2- ⁇ -globin to yield plasmid pL133. The construction protocol for plasmid pL133 is described in detail, below, and schematically illustrated in Figure 2 of the accompanying drawings.
  • the Bani-digested plasmid pHC7 DNA was then electrophoresed on a 3.5% polyacrylamide gel (29:1, acrylamide:bis-acrylamide), until the -1.25 kb Bani restriction fragment was separated from the other digestion products.
  • the region of the gel containing the -1.25 kb Bani restriction fragment was cut from the gel, placed in a test tube, and broken into small fragments.
  • One ml of extraction buffer 500 mM NH 4 0Ac, 10 mM MgOAc, 1 mM EDTA, 1 % SDS, and 10 mg/ml tRNA was added to the tube containing the fragments, and the tube was placed at 37°C overnight. Centrifugation was used to pellet the debris, and the supernatant was transferred to a new tube. The debris was washed once with 200 ⁇ l of extraction buffer; the wash supernatant was combined with the first supernatant from the overnight extraction.
  • Approximately 8 ⁇ g of the -1.25 kb Bani restriction fragment were obtained by this procedure.
  • the purified fragment was suspended in 10 ⁇ l of TE buffer and stored at -20 °C.
  • the Bani restriction fragment had to be modified by the addition of a linker to construct plasmid pSV2-HPCB.
  • the DNA fragments used in the construction of the linker were synthesized either by using a Systec 1450A DNA Synthesizer (Systec Inc., 3816 Chandler Drive, Minneapolis, MN) or an ABS 380A DNA Synthesizer (Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, CA 94404). Many DNA synthesizing instruments are known in the art and can be used to make the fragments.
  • fragments can also be conventionally prepared in substantial accordance with the procedures of Itakura et al., 1977, Science, 198:1056 and Crea et al., 1978, Proc. Nat. Acad. Sci. USA, 75:5765.
  • reaction buffer which contained 15 units (-0.5 ⁇ l) T4 polynucleotide kinase, 2 ⁇ l 10X ligase buffer, 10 ⁇ l of 500 ⁇ M ATP, and 7.5 ⁇ l of H 2 0.
  • the kinase reaction was incubated at 37°C for 30 minutes, and the reaction was terminated by incubation at 100 °C for 10 minutes.
  • reaction was chilled on ice, 2 ⁇ l of 0.2 M dithiothreitol, 2.5 ⁇ l of 5 mM ATP, and 15 units of T4 polynucleotide kinase were added to the reaction mixture and mixed, and the reaction mixture was incubated another 30 minutes at 37°C. The reaction was stopped by another 10 minute incubation at 100°C and then chilled on ice.
  • the two single strands of the DNA linker were mixed together after the kinase reaction.
  • the kinase reaction mixture was incubated at 100° C for 10 minutes in a water bath containing -150 ml of water. After this incubation, the water bath was shut off and allowed to cool to room temperature, a process taking about 3 hours. The water bath, still containing the tube of kinased DNA, was then incubated at 4°C overnight. This process annealed the single strands.
  • the linker constructed had the following structure: The linker was stored at -20 °C until use.
  • the 8 ⁇ g of -1.25 kb Bani fragment were added to and mixed with the -50 ⁇ l of linker (-500 picomoles), 1 ⁇ l of T4 DNA ligase (-5 units), 10 ⁇ l of 10X ligase buffer, and 29 ⁇ l of H 2 0, and the resulting ligation reaction was incubated at 4°C overnight. The ligation reaction was stopped by a 10 minute incubation at 65 °C. The DNA was pelleted by adding NaOAc to a final concentration of 0.3 M, adding 2 volumes of ethanol, chilling in a dry ice-ethanol bath, and then centrifuging the solution.
  • 5 ⁇ l (-50 units) of restriction enzyme Apal and 85 ⁇ l of H 2 O
  • the reaction mixture was loaded onto a 3.5% polyacrylamide gel, and the desired -1.23 kb Hindlll-Apal restriction fragment was isolated from the gel and purified. Approximately 5 ⁇ g of the desired fragment were obtained, suspended in 10 ⁇ l of TE buffer, and stored at -20 °C.
  • the -5 ⁇ g of 0.88 kb Pstl fragment were added to and mixed with -50 ⁇ l of the following linker, which was constructed on an automated DNA synthesizer: About 1 ⁇ l of T4 DNA ligase (-10 units), 10 ⁇ l 10X ligase buffer, and 29 ⁇ l H 2 O were added to the mixture of DNA, and the resulting ligation reaction was incubated at 4 °C overnight.
  • the ligation reaction was stopped by a 10 minute incubation at 65 °C. After precipitation of the ligated DNA, the DNA pellet was dissolved in 10 ⁇ l of 10X Apal reaction buffer, 5 ⁇ l (-50 units) of restriction enzyme Apal, and 85 ⁇ l of H 2 0, and the reaction was placed at 37° for two hours. The reaction was then stopped and the DNA pelleted once again.
  • plasmid pSV2gpt DNA ATCC 37145
  • 10X Hindlll reaction buffer 5 ⁇ l (-50 units) of restriction enzyme Hindlll, and 85 ⁇ l of H 2 0
  • the reaction mixture was then made 0.25 M in NaOAc, and after the addition of two volumes of ethanol and incubation in a dry ice-ethanol bath, the DNA was pelleted by centrifugation.
  • the DNA pellet was dissolved in 10 ⁇ l of 10X Bglll buffer, 5 ⁇ l (-50 units) of restriction enzyme Bglll, and 85 ⁇ l of H 2 0, and the reaction was placed at 37 °C for two hours.
  • the reaction mixture was loaded onto a 1% agarose gel, and the fragments were separated by electrophoresis.
  • the gel was stained with ethidium bromide and viewed under ultraviolet light, and the band containing the desired -5.1 kb HindIII-BgIII fragment was cut from the gel and placed in dialysis tubing, and electrophoresis was continued until the DNA was out of the agarose.
  • the buffer containing the DNA from the dialysis tubing was extracted with phenol and CHCl 3 , and then, the DNA was precipitated.
  • the pellet was resuspended in 10 ⁇ l of TE buffer and constituted 5 ⁇ g of the desired -5.1 kb HindIII-BgIII restriction fragment of plasmid pSV2gpt.
  • E. coli K12 RR1 (NRRL B-15210) cells were made competent for transformation in substantial accordance with the procedure described for E. coli K12 HB101 in Example 2B.
  • the ligated DNA prepared above was used to transform the cells, and aliquots of the transformation mix were plated on L-agar plates containing 100 ⁇ g/ml ampicillin. The plates were then incubated at 37 °C.
  • E. coli K12 RR1/pSV2-HPC8 transformants were verified by restriction enzyme analysis of their plasmid DNA. Plasmid pSV2-HPC8 DNA was prepared from the transformants in substantial accordance with the procedure of Example 1A, except that ampicillin, and not tetracycline, was used as the selective agent during culture of the cells.
  • the resulting Sail reaction mixture was incubated for 2 hours at 37°C.
  • the Hindlll-Sall-digested plasmid pSV2-HPC8 was loaded onto a 3.5% polyacrylamide gel and electrophoresed until the desired -0.29 kb Hindlll-Sall restriction fragment was separated from the other reaction products.
  • the desired fragment was isolated from the gel; about 2 ⁇ g of the fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • plasmid pSV2-HPC8 Fifty ⁇ g of plasmid pSV2-HPC8 were dissolved in 10 ⁇ l of 10X Bglll reaction buffer, 5 ⁇ l (50 units) of restriction enzyme Bglll, and 85 ⁇ l of H 2 0, and the reaction was incubated at 37°C for two hours. After the Bglll digestion, the DNA was precipitated, and the DNA pellet was dissolved in 10 ⁇ l of 10X Sail reaction buffer, 5 ⁇ l (-50 units) of restriction enzyme Sall, and 85 ⁇ l of H 2 0. The resulting Sail reaction mixture was incubated for 2 hours at 37°C.
  • the Sall-Bglll-digested plasmid pSV2-HPC8 was loaded onto a 3.5% polyacrylamide gel and electrophoresed until the desired -1.15 kb Sall-Bglll restriction fragment was separated from the other reaction products.
  • the -1.15 kb Sall-Bglll restriction fragment was isolated from the gel; about 8 ⁇ g of fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • plasmid pSV2- ⁇ -globin DNA (NRRL B-15928) were dissolved in 10 ⁇ l of 10X Hindlll reaction buffer, 5 ⁇ l (-50 units) of restriction enzyme Hindlll, and 85 ⁇ l of H 2 0, and the reaction was placed at 37 °C for 2 hours. The reaction mixture was then made 0.25 M in NaOAc, and after the addition of two volumes of ethanol and incubation in a dry ice-ethanol bath, the DNA was pelleted by centrifugation.
  • Hindlll-digested plasmid pSV2- ⁇ -globin was dissolved in 10 ⁇ l of 10X Bglll buffer, 5 ⁇ l (-50 units) of restriction enzyme Bglll, and 85 ⁇ l of H 2 0, and the reaction was placed at 37°C for two hours. After the Bglll digestion, the reaction mixture was loaded onto a 1% agarose gel, and the fragments were separated by electrophoresis. The desired -4.2 kb HindIII-BgIII restriction fragment was isolated from the gel; about 5 ⁇ g of the desired fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • the ligated DNA constituted the desired plasmid pL133; a restriction site and function map of plasmid pL133 is presented in Figure 2 of the accompanying drawings.
  • the ligated DNA was used to transform E. coli K12 RR1, and the desired E. coli K12 RR1/pL133 transformants were identified by their ampicillin-resistant phenotype and by restriction enzyme analysis of their plasmid DNA.
  • plasmid pBLcat DNA About 20 ⁇ g of plasmid pBLcat DNA were dissolved in 10 ⁇ l of 10X Hindlll buffer and 80 ⁇ l of H 2 0. About 10 ⁇ l (-100 units) of restriction enzyme Hindlll were added to the solution of plasmid pBLcat DNA, and the resulting reaction was incubated at 37 °C for 2 hours. The Hindlll-digested plasmid pBLcat DNA was loaded onto an agarose gel and electrophoresed until the -0.87 kb Hindlll restriction fragment that comprises the BK enhancer and Ad2 late promoter was separated from the other digestion products; then, the -0.87 kb fragment was isolated, purified, and prepared for ligation. About 2 ⁇ g of the desired fragment were obtained and dissolved in 5 ⁇ l of TE buffer.
  • plasmid pL133 DNA About 1.5 ⁇ g of plasmid pL133 DNA were dissolved in 2 ⁇ l of 10X Hindlll buffer and 16 ⁇ l of H 2 0. About 1 ⁇ l (-10 units) of restriction enzyme Hindlll was added to the solution of DNA, and the resulting reaction was incubated at 37 ° C for 2 hours. The DNA was then diluted to 100 ⁇ l with TE buffer and treated with -0.06 units of calf-intestinal alkaline phosphatase, and the resulting reaction was incubated at 37 ° C for 30 minutes.
  • the Hindlll-digested plasmid pL133 DNA was then extracted twice with phenol and once with chloroform, precipitated with ethanol, and resuspended in 10 ⁇ l of TE buffer.
  • the ligated DNA was used to transform E. coli K12 HB101 in substantial accordance with the procedure of Example 2B.
  • the transformed cells were plated on L-agar plates containing ampicillin, and the plasmid DNA of the ampicillin-resistant transformants was examined by restriction enzyme analysis to identify the E. coli K12 HB101/pLPC transformants.
  • the -0.87 kb Hindlll restriction fragment that encodes the BK enhancer and Ad2 late promoter could insert into Hindlll-digested plasmid pL133 in one of two orientations, only one of which yields plasmid pLPC.
  • a restriction site and function map of plasmid pLPC is presented in Figure 1, Part D, of the accompanying drawings.
  • Plasmid pLPC-167G was constructed in substantial accordance with the site-specific mutagenesis and other construction protocols used in the construction of plasmid pLAPC, as described in Example 1. Buffers and annealing conditions used in the construction of plasmid pLPC-167G, however, were as described by Zoller and Smith, 1984, DNA 3:479-489.
  • phage M13mp18-HE1 were subjected to site-specific mutagenesis using the mutagenizing oligonucleotide depicted below:
  • Plasmid pLPC-167G proceeded in a manner analogous to the construction of plasmid pLAPC, set forth in Example 1 C.
  • plasmid pLAPC was constructed using two restriction fragments originating from plasmid pLPC.
  • these same two fragments were instead obtained from plasmid pLAPC.
  • plasmids pLPC and pLPC-167G are very close to the same size, it would have been difficult to distinguish "parentals" (plasmid pLPC) from plasmid pLPC-167G. These parentals could be present, despite the purification of the fragments used in the ligation, due to a variety of factors. However, because plasmid pLAPC is smaller than plasmid pLPC-167G, by obtaining the two fragments from plasmid pLAPC, one could readily distinguish parentals (plasmid pLAPC) from the desired plasmid pLPC-167G.
  • plasmid pLPC-167G the -0.7 kb Sstl-Sall restriction fragment of phage M13mp18-HE4 was ligated to the -3.76 kb EcoRI-Sall restriction fragment of plasmid pLAPC and the -2.0 kb EcoRI-Sstl restriction fragment of plasmid pLAPC.
  • the ligated DNA constituted the desired plasmid pLPC-167G, which was transformed into E. coli K12 RV308.
  • the resulting E. coli K12 RV308/pLPC-167G transformants were used to obtain a large-scale preparation of plasmid pLPC-167G DNA for use in transformations of eukaryotic cells.
  • Plasmid pLPC-167F was constructed in substantial accordance with the site-specific mutagenesis and other construction protocols used in the construction of plasmid pLAPC, as described in Example 1. Buffers and annealing conditions used in the construction of plasmid pLPC-167F, however, were as described by Zoller and Smith, 1984, DNA 3:479-489.
  • phage M13mp18-HE1 were subjected to site-specific mutagenesis using the mutagenizing oligonucleotide depicted below:
  • Plasmid pLPC-167F proceeded in a manner analogous to the construction of plasmid pLAPC, set forth in Example 1 C.
  • plasmid pLAPC was constructed using two restriction fragments originating from plasmid pLPC.
  • these same two fragments were instead obtained from plasmid pLAPC.
  • plasmids pLPC and pLPC-167F are very close to the same size, it would have been difficult to distinguish "parentals" (plasmid pLPC) from plasmid pLPC-167F.
  • plasmid pLAPC is smaller than plasmid pLPC-167F, by obtaining the two fragments from plasmid pLAPC, one could readily distinguish parentals (plasmid pLAPC) from the desired plasmid pLPC-167F.
  • plasmid pLPC-167F the -0.7 kb Sstl-Sall restriction fragment of phage M13mp18-HE5 was ligated to the -3.76 kb EcoRI-Sall restriction fragment of plasmid pLAPC and the -2.0 kb EcoRI-Sstl restriction fragment of plasmid pLAPC.
  • the ligated DNA constituted the desired plasmid pLPC-167F, which was transformed into E. coli K12 RV308.
  • the resulting E. coli K12 RV308/pLPC-167F transformants were used to obtain a large-scale preparation of plasmid pLPC-167F DNA for use in transformations of eukaryotic cells.
  • Human Embryonic Kidney Cell Line 293 is available from the American Type Culture Collection under the accession number ATCC CRL 1573.
  • the adenovirus-transformed Syrian hamster cell line AV12 is also available from the American Type Culture Collection under the accession number ATCC CRL 9595.
  • the transformation procedure described below refers to 293 cells as the host cell line; however, the procedure is generally applicable to most eukaryotic cell lines, including the AV12 cell line, and to the expression vectors of the invention.
  • 293 cells are obtained from the ATCC under the accession number CRL 1573 in a 25 mm 2 flask containing a confluent monolayer of about 5.5 x 10 6 cells in Eagle's Minimum Essential Medium (Gibco) with 10% heat-inactivated horse serum. The flask is incubated at 37 °C; medium is changed twice weekly. Media is composed of DMEM (Gibco) supplemented with 10% fetal calf serum, 50 ⁇ g/ml gentamicin, and 10 ⁇ g/ml AquaMEPHYTONO phytonadione vitamin K 1 (Merck Sharp and Dohme, Merck and Co., Inc., West Point, PA 19486).
  • the cells are subcultured by removing the medium, rinsing with Hank's Balanced Salts solution (Gibco), adding 0.25% trypsin (containing 0.2 g/L EDTA) for 1-2 minutes, rinsing with fresh medium, aspirating, and dispensing into new flasks at a subcultivation ratio of 1:5 or 1:10.
  • Hank's Balanced Salts solution Gibco
  • trypsin containing 0.2 g/L EDTA
  • plasmid pLPC-167F or pLPC-167G transformations usually two plasmids are used, plasmid pLPC-167F or pLPC-167G and a plasmid that contains a selectable marker, as discussed below) and 250 mM CaCI 2 .
  • 2X HBSS is prepared containing 280 mM NaCI, 50 mM Hepes, and 1.5 mM sodium phosphate, with the pH adjusted to 7.05-7.15.
  • the 2X DNA-CaC1 2 solution is added dropwise to an equal volume of sterile 2X HBSS.
  • a one ml sterile plastic pipette with a cotton plug is inserted into the mixing tube that contains the 2X HBSS, and bubbles are introduced by blowing while the DNA is being added.
  • the calcium-phosphate-DNA precipitate is allowed to form without agitation for 30-45 minutes at room temperature.
  • the transformation procedure utilizes a mixture of plasmids: the expression vector of the present invention that lacks a selectable marker; and an expression vector that comprises a selectable marker that functions in eukaryotic cells.
  • Plasmid pSV2-hyg confers resistance to hygromycin B to eukaryotic host cells.
  • This co-transformation technique allows for the selection of cells that contain the plasmid with the selectable marker. These cells are further examined to identify cells that comprise both of the transforming plasmids.
  • the present invention also comprises expression vectors that contain a selectable marker for eukaryotic cells and thus do not require use of the cotransformation technique.
  • hygromycin B is added to the growth medium to a final concentration of about 200 ug/ml. The cells are then incubated at 37 °C for 2-4 weeks with medium changes at 3 to 4 day intervals. The resulting hygromycin-resistant colonies are transferred to individual culture flasks for characterization. Plasmid pSV2-neo confers resistance to neomycin (G418 is also used in place of neomycin), and selection of G418-resistant colonies is performed in substantial accordance with the selection procedure for hygromycin-resistant cells, except that G418 is added to a final concentration of 400 ug/ml.
  • dhfr dihydrofolate reductase
  • dhfr-mtx methotrexate resistance-conferring derivative of the dhfr gene
  • Cell lines that do lack a functional dhfr gene and are transformed with dhfr-containing plasmids can be selected for on the basis of the dhfr + phenotype.
  • dhfr as a selectable and amplifiable marker in dhfr-producing cells
  • the present invention is not limited by the selectable marker used on expression vectors.
  • amplifiable markers such as metallothionein genes, adenosine deaminase genes, or members of the multigene resistance family, exemplified by the P-glyco-protein gene, can be utilized.
  • Transformation of the 293 and AV12 cell lines with a mixture of plasmid pLPC-167F or pLPC-167G and a hygromycin resistance-conferring vector and subsequent selection for hygromycin-resistant cells yielded a number of transformants.
  • These transformants are analyzed, as described in Example 6, to determine which hygromycin-resistant cells contained plasmid pLPC-167F or pLPC-167G.
  • the hygromycin-resistant transformants obtained in Example 5 are grown on 100 mm 2 tissue culture dishes at a density of several hundred cell clones per tissue culture dish. The media is decanted, and the cells are rinsed twice with 5 ml aliquots of Hank's Balanced salt solution (Gibco). A solution of sterile 0.45% agar (Sigma Type 4 agarose, catalogue #A3643, Sigma Chemical Co., P.O. Box 14508, St.
  • Louis, MO 63178 is prepared by mixing 1 ml of 1.8% agar (47 °C) with 3 ml of Dulbecco's Modified Eagle's (DME) Salts (Gibco) (37 ° C), and 2 ml of this 0.45% agar solution are layered over the cells.
  • DME Dulbecco's Modified Eagle's
  • the cells are overlayed with 8 ml of a mixture containing 2 ml of 1.8% agar (47 °C), 2 ml of DME salts (37 °C), and 4 ml of DME salts with 20% fetal bovine serum (37 °C). The cells are then placed in a 37 ° C incubator.
  • Polyclonal antibody can be prepared by the procedure disclosed in Structural Concepts in Immunology and Immunochemistry by E.A. Kabat, published in 1968 by Holt, Rhinehart, and Winston.
  • Monoclonal antibody which is also suitable for use in the assay, can be prepared as disclosed in Kohler and Milstein, 1975, Nature, 256:495, or as disclosed in U.S. Patent No. 4,696,895; EPO Pub. No. 205046; Laurell et al., 1985, FEBS 191 (1):75; Suzuki et al., 1985, J. Biochem.
  • the avidin D and biotinylated horse radish peroxidase (HRP) used in the assay are obtained in a Vectastain TM kit (Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010). Biotin is also obtained from Vector Laboratories, Inc.
  • the filters are rinsed four times with PBS at 4 ° C. Then, avidin D and biotinylated horse radish peroxidase are prepared and added as per the manufacturer's instructions in the VectastainTM (Vector Laboratories) kit. The filters are incubated with the HRP-conjugated avidin D for 1 hour at 4°C (longer incubation times, i.e., overnight, can be used when small amounts of protein are being secreted); then, the filters are rinsed four times with PBS at 4°C.
  • HRP color-development reagent (4-chloro-1-napthol, Sigma) dissolved in ice-cold 100% methanol are added to 50 ml of PBS and 30 ⁇ l of 30% H 2 0 2 . This mixture is added to the nitrocellulose filters, which are incubated at room temperature until the color develops. Colonies secreting the most human protein C zymogen of the invention will be indicated on the filters not only by earliest appearance of the color but also by darker spots on the filter.
  • the filters are again realigned with the original plates to determine which colonies are associated with which spots on the filter.
  • the colonies secreting the most human protein C zymogen of the invention are then selected and used for production of the zymogen.
  • the above assay is merely illustrative of the method of identifying high secreting cell lines.
  • a variety of assay procedures can be successfully employed in the method. For instance, a double-antibody reaction can be employed in which the biotinylated goat anti protein C antibody is replaced with a goat anti-protein C antibody (IgG) and a biotinylated anti-goat IgG antibody.
  • IgG goat anti-protein C antibody

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Claims (25)

1. DNA Verbindung, die eine kodierende Sequenz für ein Protein umfaßt, das vom Aminoterminus bis zum Carboxyterminus umfaßt:
a) ein Signalpeptid und Propeptid eines gamma-carboxylierten, sekretierten Proteins,
b) die leichte Kette von humanem Protein C,
c) das Dipeptid Lysin-Arginin, Arginin-Lysin, Lysin-Lysin oder Arginin-Arginin, und
d) folgende Aminosäuresequenz
Figure imgb0037
worin R1 für PHE, GLY, TYR oder TRP steht, R2 für VAL oder PRO steht, R3 für ASP oder ASN steht, ARG für Arginin steht, ASN für Asparagin steht, ASP für Asparaginsäure steht, -COOH für den Carboxyterminus steht, CYS für Cystein steht, GLN für Glutamin steht, GLU für Glutaminsäure steht, GLY für Glycin steht, HIS für Histidin steht, ILE für Isoleucin steht, LEU für Leucin steht, LYS für Lysin steht, MET für Methionin steht, PHE für Phenylalanin steht, PRO für Prolin steht, SER für Serin steht, THR für Threonin steht, TRP für Tryptophan steht, TYR für Tyrosin steht und VAL für Valin steht.
2. DNA Verbindung nach Anspruch 1, worin das Signalpeptid und das Propeptid das Signalpeptid und das Propeptid von unreifem humanem Protein C sind.
3. DNA Verbindung nach Anspruch 1 oder 2, worin das Dipeptid Lysin-Arginin ist.
4. DNA Verbindung nach Anspruch 3, worin das durch die DNA kodierte Polypeptid folgendes ist
Figure imgb0038
Figure imgb0039
worin -H2N für den Aminoterminus steht, R1 für PHE, GLY, TYR oder TRP steht, R2 für PRO oder VAL steht und R3 für ASP oder ASN steht.
5. Rekombinanter DNA Expressionsvektor, der die DNA Verbindung nach Anspruch 1, 2, 3 oder 4 umfaßt.
6. Vektor nach Anspruch 5, worin R1 für PHE steht, R2 für PRO steht und R3 für ASP steht.
7. Vektor nach Anspruch 6, der das wie in Beispiel 4 beschrieben hergestellte Plasmid pLPC-167F ist.
8. Vektor nach Anspruch 5, worin R1 für GLY steht, R2 für PRO steht und R3 für ASP steht.
9. Vektor nach Anspruch 8, der das wie in Beispiel 3 beschrieben hergestellte Plasmid pLPC-167G ist.
10. Eukaryontische Wirtszelle, die mit einem Vektor nach Anspruch 5 transformiert ist.
11. Eukaryontische Wirtszelle nach Anspruch 10, die 293/pLPC-167F ist, welche durch Transformation der humanen embryonalen Nierenzellinie 293 mit dem Plasmid pLPC-167F nach Anspruch 7 hergestellt ist.
12. Eukaryontische Wirtszelle nach Anspruch 10, die 293/pLPC-167G ist, welche durch Transformation der humanen embryonalen Nierenzellinie 293 mit dem Plasmid pLPC-167G nach Anspruch 9 hergestellt ist.
13. Eukaryontische Wirtszelle nach Anspruch 10, die AV12/pLPC-167F ist, welche durch Transformation der Adenovirus-transformierten syrischen Hamsterzellinie AV12 mit dem Plasmid pLPC-167F nach Anspruch 7 hergestellt ist.
14. Eukaryontische Wirtszelle nach Anspruch 10, die AV12/pLPC-167G ist, welche durch Transformation der Adenovirus-transformierten syrischen Hamsterzellinie AV12 mit dem Plasmid pLPC-167G nach Anspruch 9 hergestellt ist.
15. Verfahren zur rekombinanten Herstellung einer Zymogenform von humanem Protein C durch Sekretion aus einer eukaryontischen Wirtszelle, gekennzeichnet durch
(A) Transformation einer eukaryontischen Wirtszelle mit einem rekombinanten DNA Vektor, der umfaßt
(i) eine DNA Sequenz, die eine Aminosäuresequenz kodiert, welche vom Aminoterminus zum Carboxyterminus umfaßt
a) ein Signalpeptid und ein Propeptid eines gamma-carboxylierten, sekretierten Proteins,
b) die leichte Kette von humanem Protein C,
c) das Dipeptid LYS-ARG, ARG-LYS, LYS-LYS oder ARG-ARG, und
d) folgende Aminosäuresequenz
Figure imgb0040
worin R1 für PHE, GLY, TYR oder TRP steht, R2 für VAL oder PRO steht, R3 für ASP oder ASN steht, ARG für Arginin steht, ASN für Asparagin steht, ASP für Asparaginsäure steht, -COOH für den Carboxyterminus steht, CYS für Cystein steht, GLN für Glutamin steht, GLU für Glutaminsäure steht, GLY für Glycin steht, HIS für Histidin steht, ILE für Isoleucin steht, LEU für Leucin steht, LYS für Lysin steht, MET für Methionin steht, PHE für Phenylalanin steht, PRO für Prolin steht, SER für Serin steht, THR für Threonin steht, TRP für Tryptophan steht, TYR für Tyrosin steht und VAL für Valin steht, und
(ii) einen Promotor, der zur Expressionssteuerung der DNA Sequenz positioniert ist, und
(B) Kultivierung der in Schritt (A) transformierten Wirtszelle unter Bedingungen, die die Expression der DNA Sequenz erlauben.
16. Verfahren nach Anspruch 15, worin der rekombinante DNA Expressionsvektor das Plasmid pLPC-167F nach Anspruch 7 ist.
17. Verfahren nach Anspruch 15, worin der rekombinante DNA Expressionsvektor das Plasmid pLPC-167G nach Anspruch 9 ist.
18. Verfahren nach Anspruch 15, worin die Wirtszelle die 293- oder AV12-Wirtszelle ist.
19. Verfahren nach Anspruch 18, worin die in Schritt (B) kultivierte Wirtszelle die 293/pLPC-167F, 293/pLPC-167G, AV12/pLPC-167F oder AV12/pLPC-167G Wirtszelle nach den Ansprüchen 11 bis 14 ist.
20. Protein C Zymogen mit der folgenden Aminosäuresequenz
Figure imgb0041
Figure imgb0042
worin H2N für den Aminoterminus steht, R1 für PHE, GLY, TYR oder TRP steht, R2 für VAL oder PRO steht, R3 für ASP oder ASN steht, ARG für Arginin steht, ASN für Asparagin steht, ASP für Asparaginsäure steht, -COOH für den Carboxyterminus steht, CYS für Cystein steht, GLN für Glutamin steht, GLU für Glutaminsäure steht, GLY für Glycin steht, HIS für Histidin steht, ILE für Isoleucin steht, LEU für Leucin steht, LYS für Lysin steht, MET für Methionin steht, PHE für Phenylalanin steht, PRO für Prolin steht, SER für Serin steht, THR für Threonin steht, TRP für Tryptophan steht, TYR für Tyrosin steht und VAL für Valin steht.
21. Zymogen nach Anspruch 20, worin R1 für PHE steht, R2 für PRO steht und R3 für ASP steht.
22. Zymogen nach Anspruch 20, worin R1 für GLY steht, R2 für PRO steht und R3 für ASP steht.
23. Aktiviertes Protein C Molekül, das die leichte Kette von aktiviertem humanem Protein C und die folgende schwere Kette umfaßt
Figure imgb0043
worin ALA für Alanin steht, ARG für Arginin steht, ASN für Asparagin steht, ASP für Asparaginsäure steht, -COOH für den Carboxyterminus steht, CYS für Cystein steht, GLN für Glutamin steht, GLU für Glutaminsäure steht, GLY für Glycin steht, H2N für den Aminoterminus steht, HIS für Histidin steht, ILE für Isoleucin steht, LEU für Leucin steht, LYS für Lysin steht, MET für Methionin steht, PHE für Phenylalanin steht, PRO für Prolin steht, SER für Serin steht, THR für Threonin steht, TRP für Tryptophan steht, TYR für Tyrosin steht und VAL für Valin steht.
24. Pharmazeutische Formulierung, die das Zymogen nach Anspruch 20 zusammen mit einem pharmazeutisch annehmbaren Träger hierfür umfaßt.
25. Pharmazeutische Formulierung, die das aktivierte Protein C nach Anspruch 23 zusammen mit einem pharmazeutisch annehmbaren Träger hierfür umfaßt.
EP88312201A 1987-12-28 1988-12-22 Vektoren und Verbindungen zur Expression von Zymogen-Formen von menschlichem Protein C Expired - Lifetime EP0323149B1 (de)

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EP0380508A4 (en) * 1987-05-18 1991-04-03 Integrated Genetics, Inc. Improved protein c molecules and method for making and activating same
US4992373A (en) * 1987-12-04 1991-02-12 Eli Lilly And Company Vectors and compounds for direct expression of activated human protein C
GB8927722D0 (en) * 1989-12-07 1990-02-07 British Bio Technology Proteins and nucleic acids
EP0506821B1 (de) * 1989-12-29 1998-02-04 Zymogenetics, Inc. Hybrides protein c
US5358932A (en) * 1989-12-29 1994-10-25 Zymogenetics, Inc. Hybrid protein C
US5270178A (en) * 1990-02-23 1993-12-14 Eli Lilly And Company Vectors and compounds for expression of zymogen forms of human protein C
IL97311A0 (en) * 1990-02-23 1992-05-25 Lilly Co Eli Vectors and compounds for expression of glycosylation mutants of human protein c
MY110664A (en) * 1992-05-21 1999-01-30 Lilly Co Eli Protein c derivatives
US5618714A (en) * 1993-12-15 1997-04-08 Eli Lilly And Company Methods for producing protein C
RU2214833C2 (ru) * 1994-10-24 2003-10-27 Ново Нордиск Хелс Кеар Аг Модифицированный фактор vii
JP2003514545A (ja) * 1999-11-19 2003-04-22 イーライ・リリー・アンド・カンパニー プロテインｃ誘導体
PL1781782T3 (pl) * 2004-08-17 2010-10-29 Csl Behring Gmbh Zmodyfikowane polipeptydy zależne od witaminy K

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